Dental caries is recognized as the most prevalent infectious disease on earth and its treatment continues to have a major economic impact on developed countries. Streptococcus mutans is the principal etiologic agent of this disease and the unique ability of this organism to metabolize dietary sucrose is known to be crucial to virulence. Especially important in this regard is the production of extracellular glucans from sucrose by multiple glucosyltransferase enzymes. In the present application we propose to extend our studies utilizing molecular genetics to gain a more complete understanding of the biochemical genetic basis of glucan synthesis and other sucrose-related virulence traits. We shall complete our construction of S. mutans genomic libraries using lambda phage cloning and employ a variety of immunological and physiological screening techniques to identify the genetic determinants specifying glucan synthesis, dextranase and sucrase activity. Two such genes have already been identified, one of which seems involved in water-insoluble glucan synthesis. Using all of these cloned genes as probes in various types of hybridization strategies, we shall test the hypothesis that certain virulence related genes governing sucrose metabolism are linked in S. mutans. We shall construct defective copies of all of these genes in E. coli and introduce them back into S. mutans by transformation. Strains carrying single, double and multiple mutations constructed by this methodology will be studied to define the individual and collective roles of these determinants in sucrose-related glucan synthesis, adherence, cell aggregation, acid production and virulence in animals. Finally, we shall take advantage of our cloned glucosyltransferase genes as DNA probes to study the possibility that genetic expression of these loci is controlled. Specifically, because glucan synthesis appears to shut down spontaneously in cells cultured in vitro, it is our hypothesis that these genes are regulated by a control mechanism involving genomic rearrangement reminiscent of phase variation seen in other pathogenic bacteria. We shall examine the local genomic environment of the glucosyltransferase genes using appropriate DNA probes in order to test this hypothesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DE004224-21
Application #
2128952
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-05-01
Project End
1996-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
21
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Fletcher, H M; Schenkein, H A; Macrina, F L (1994) Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis. Infect Immun 62:4279-86
Munro, C L; Macrina, F L (1993) Sucrose-derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis. Mol Microbiol 8:133-42
Munro, C; Michalek, S M; Macrina, F L (1991) Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange. Infect Immun 59:2316-23
Krah 3rd, E R; Macrina, F L (1991) Identification of a region that influences host range of the streptococcal conjugative plasmid pIP501. Plasmid 25:64-9
Dertzbaugh, M T; Macrina, F L (1990) Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide. Infect Immun 58:1509-13
Dertzbaugh, M T; Peterson, D L; Macrina, F L (1990) Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein. Infect Immun 58:70-9
Krah 3rd, E R; Macrina, F L (1989) Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501. J Bacteriol 171:6005-12
Dertzbaugh, M T; Macrina, F L (1989) Plasmid vectors for constructing translational fusions to the B subunit of cholera toxin. Gene 82:335-42
Schroeder, V A; Michalek, S M; Macrina, F L (1989) Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403. Infect Immun 57:3560-9